Fibroblast growth factor receptor 3 isoforms: Novel therapeutic targets for hepatocellular carcinoma?

UNLABELLED: Fibroblast growth factor receptors (FGFRs) are frequently up-regulated in subsets of hepatocellular carcinoma (HCC). Here, we provide mechanistic insight that FGFR3 splice variants IIIb and IIIc impact considerably on the malignant phenotype of HCC cells. The occurrence of FGFR3 variants was analyzed in human HCC samples. In hepatoma/hepatocarcinoma cell lines, FGFR3 isoforms were overexpressed by lentiviral constructs or down-modulated by small interfering RNA (siRNA; affecting FGFR3-IIIb and -IIIc) or an adenoviral kinase-dead FGFR3-IIIc construct (kdFGFR3). Elevated levels of FGFR3-IIIb and/or -IIIc were found in 53% of HCC cases. FGFR3-IIIb overexpression occurred significantly more often in primary tumors of large (pT2-4) than of small size (pT1). Furthermore, one or both isoforms were enhanced mostly in cases with early tumor infiltration and/or recurrence at the time of surgery or follow-up examinations. In hepatoma/hepatocarcinoma cells, up-regulated FGFR3-IIIb conferred an enhanced capability for proliferation. Both FGFR3-IIIb and FGFR3-IIIc suppressed apoptotic activity, enhanced clonogenic growth, and induced disintegration of the blood/lymph endothelium. The tumorigenicity of cells in severe combined immunodeficiency mice was augmented to a larger degree by variant IIIb than by IIIc. Conversely, siRNA targeting FGFR3 and kdFGFR3 reduced clonogenicity, anchorage-independent growth, and disintegration of the blood/lymph endothelium in vitro. Furthermore, kdFGFR3 strongly attenuated tumor formation in vivo. CONCLUSIONS: Deregulated FGFR3 variants exhibit specific effects in the malignant progression of HCC cells. Accordingly, blockade of FGFR3-mediated signaling may be a promising therapeutic approach to antagonize growth and malignant behavior of HCC cells.

TP53 germline mutation may affect response to anticancer treatments: analysis of an intensively treated Li-Fraumeni family.

Li-Fraumeni syndrome (LFS) is a rare autosomal dominant inherited disorder associated with the occurrence of a wide spectrum of early-onset malignancies, the most prevalent being breast cancer and sarcoma. The presence of TP53 germline mutations in the majority of LFS patients suggests a genetic basis for the cancer predisposition. No special recommendations for the treatment of LFS patients have been made to date, except that of minimizing radiation. We hypothesized that TP53 germline mutations may be associated not only with cancer predisposition, but also with lack of response to chemo- and radiotherapy. Here, we present an Austrian LFS family whose members were intensively treated with chemo- and radiotherapy due to cancers that occurred at a predominantly young age, including eight breast cancers in six patients. Material from seven family members was screened for p53 mutation by Sanger sequencing and immunohistochemistry. A rare missense mutation in the tetramerization domain of exon 10 of the TP53 gene was found to segregate with malignant disease in this family. Lack of response to various chemotherapies and radiotherapy could be ascertained by histopathology of surgical specimens after neoadjuvant treatment, by cancer relapse occurring while receiving adjuvant systemic treatment and by the occurrence of second primaries in areas of adjuvant radiation. Our observations suggest that current standards of cancer treatment may not be valid for patients with LFS. In patients with TP53 germline mutation, cytotoxic treatment may bear not only the risk of tumor induction but also the risk of treatment failure.

Genetic detection of lymph node micrometastases: a selection criterion for liver transplantation in patients with liver metastases after colorectal cancer.

BACKGROUND: Liver transplantation for nonresectable liver metastases from colorectal cancer was abandoned in 1994 on account of high recurrence rates. The aim of this study was to investigate whether the genetic detection of micrometastases in histologically negative lymph nodes of the primary colon cancer could be applied to select patients for liver transplantation. METHODS: We analyzed 21 patients with colorectal cancer who had undergone liver transplantation between 1983 and 1994 for liver metastases. Eleven patients were histologically lymph node negative at the time of surgery; ten patients with lymph node metastases served as control group. DNA sequencing was used to screen tumor material for p53 and K-ras mutations. Mutant allele-specific amplification (MASA) was then used to search for micrometastases in DNA from regional lymph nodes of the primary colorectal cancer. RESULTS: p53 and K-ras mutations were detected in 12 (57%) and 3 (14%) of 21 patients in the colorectal cancer, respectively. The mutations were confirmed in the corresponding liver metastases. Of 11 patients with histologically negative lymph nodes, nine were eligible for MASA due to presence of p53 or K-ras mutation. MASA revealed six of nine patients to be genetically positive for micrometastases. Three patients were both genetically and histologically negative. These three patients showed a significantly longer overall survival (P = 0.011) of 4, 5, and 20 years, respectively. CONCLUSIONS: We conclude that the genetic detection of micrometastases by MASA could be a powerful prognostic indicator for selecting patients with colorectal liver metastases who could benefit from liver transplantation.

TP53 Mutational Status and Prediction of Benefit from Adjuvant 5-Fluorouracil in Stage III Colon Cancer Patients.

We investigated the hypothesis that the varying treatment efficacy of adjuvant 5-fluorouracil (5FU) in stage III colon cancer is linked to the TP53 mutational status. ABCSG-90 was a prospective randomized trial in which effect of adjuvant 5FU was studied in stage III colon cancer patients. Tumor material of 70% of these patients (389/572) was available for analysis of the biomarker TP53 using a TP53-gene-specific Sanger sequencing protocol. Median follow-up was 88 months. TP53 mutation frequency was 33%. A significant interaction between TP53 status, outcomes and nodal category was found (P = 0.0095). In the N1 category, TP53 wildtype patients had significantly better overall survival than TP53 mutated (81.0% vs. 62.0% overall survival at 5 years; HR = 2.131; 95% CI: 1.344-3.378; P = 0.0010). In the N2 category, the TP53 status did not affect survival (P = 0.4992). In TP53 wildtype patients, the prognostic significance of N category was significantly enhanced (P = 0.0002). In TP53 mutated patients, survival curves of N1 and N2 patients overlapped and nodal category was no longer prognostic. The biomarker TP53 independently predicted effect of adjuvant 5FU in N1 colon cancer patients. TP53 was not predictive in N2 patients, in whom 5FU is known to have no effect.

Fatigue behavior of the resin-resin bond of partially replaced resin-based composite restorations.

PURPOSES: (1) To evaluate different pre-treatment modes for partial resin-based composite repair using a shear bond strength fatigue design on aged specimens, and (2) to investigate the effect of these pre-treatments on aged and partially replaced Class II resin-based composite restorations. MATERIALS AND METHODS: (Study 1) 150 composite discs (Tetric Ceram) were light-cured, secondary cured, and stored in water for 365 days. The specimens were randomly assigned to five pretreatment groups (n=30): (1) Roughening with a finishing diamond bur + dentin bonding system (DBS, Syntac Classic), (2) Roughening with a silicon carbide bur + DBS, (3) Roughening with a silicon carbide bur + DBS + Tetric Flow, (4) silica-coating (CoJet) + DBS, and (5) silica-coating + dentin bonding agent + Tetric Flow. The disks were restored with fresh repair composite cylinders and after 24-hour water storage, initial bond strengths (n=10) and fatigue bond strengths (n=20; 5,000 cycles in shear mode) were evaluated and fracture modes determined under a SEM. (Study 2): 40 Class II restorations (EBS Multi/Pertac II) were placed in extracted human third molars. The specimens were stored for 365 days and then replaced partially by removing the proximal box and leaving the occlusal part. The partial repairs (Pertac II) were placed after identical pre-treatment modes A-E (n=8). After thermomechanical loading (100,000 x 50 N, simultaneously x2,500 at 5 degrees C/55 degrees C), marginal quality between old and fresh composite was evaluated by SEM at x200 magnification and dye penetration was observed by light microscopy under x50 magnification. RESULTS: Initial repair bond strengths were significantly higher for the groups with additional application of flowable composite compared to composite only (P<0.05; Mann Whitney U test). Diamond finishing revealed the lowest bond strengths. Fatigue bond strengths exhibited the worst results for Group 1 as well, but were similar for Groups 2-5. In the Class II partial repair simulation, Groups 3 and 5 with additional flowable lining exhibited significantly better marginal quality and significantly lower leakage, whereas the other groups revealed up to 100% separation between aged and freshly applied composite.

The biomarker TP53 divides patients with neoadjuvantly treated esophageal cancer into 2 subgroups with markedly different outcomes. A p53 Research Group study.

BACKGROUND: Fluorouracil and cisplatin have been used most frequently as neoadjuvant therapy for esophageal cancer. Both drugs are believed to act via a p53-dependent apoptosis pathway. The TP53 gene is frequently mutated in esophageal cancer. OBJECTIVE: To test the value of TP53 as a biomarker prognosing outcome in patients with neoadjuvantly treated esophageal cancer. PATIENTS AND METHODS: The investigation included 36 patients with primary operable esophageal cancer who were treated neoadjuvantly with cisplatin and fluorouracil. The TP53 genotype was assessed from paraffin-embedded diagnostic tumor biopsies using a standardized gene-specific TP53 sequencing protocol (mark53 kit; mark53 Ltd, Vienna, Austria). RESULTS: Mutations in the TP53 gene were present in 50% of tumors. Two-year overall survival rates were 55.6% in patients with a normal TP53 marker status, compared with 16.7% in those with a mutant TP53 gene. In patients with normal TP53, neoadjuvant treatment resulted in significant advantages in terms of tumor-associated survival (P=.0049) and overall survival (P=.0304) compared with those with mutant TP53. The median tumor-associated survival was 34.2 months for patients with normal TP53, compared with 8.9 months for those with mutant TP53. The latter had a 3-fold higher risk of dying (hazard ratio, 3.01; 95% confidence interval, 1.359-6.86). CONCLUSIONS: The biomarker TP53 divides esophageal cancer patients into 2 categories with markedly different outcomes: patients with a normal TP53 marker status may experience notable benefits from neoadjuvant chemotherapy with cisplatin/fluorouracil, whereas those with a mutant TP53 marker status appear to be at risk for lack of response.

Strategy for prevention of cancers of the esophagus.

The following, from the 12th OESO World Conference: Cancers of the Esophagus, includes commentaries on the animal reflux-inflammation models for Barrett's esophagus and esophageal adenocarcinoma; genomic/epigenomic analyses; eflornithine-based combinations; the molecular derangements that promote neoplastic transformation; the role of COX-2 inhibitors, proton pump inhibitors, and phase II trials in Barrett's adenocarcinoma; statins in chemoprevention and treatment of esophageal cancer; and biomarkers as potential targets in Barrett's adenocarcinoma.

Growing clinical evidence for the interaction of the p53 genotype and response to induction chemotherapy in advanced non-small cell lung cancer.

OBJECTIVE: The objective of this study is to establish clinical evidence that the p53 genotype can serve as a predictive marker for response to cisplatin-based induction therapy. METHODS: Patients with advanced non-small cell lung cancer who had received neoadjuvant chemotherapy in the context of a prospective phase II trial were analyzed for the p53 genotype of their tumors. Response to induction therapy was then correlated to the p53 genotype as assessed by complete direct DNA sequencing. Patients had received 3 cycles of cisplatin and etoposide, and 1 cycle of simultaneous radiochemotherapy. All 3 treatment components mediate their cytotoxic effect through induction of apoptosis, which is suggested to require an intact p53 gene. In addition, the results from a previously published hypothesis-finding study are updated to demonstrate the consistency of clinical results and summarize currently available clinical evidence. RESULTS: In the phase II trial, 35 patients underwent resection after induction chemotherapy, allowing a pathohistologic response assessment. The presence of a mutant p53 genotype was highly indicative of resistance to induction chemotherapy (P < .002). The sensitivity of a mutant p53 genotype to identify nonresponders was 94% (71.3-99.9 confidence interval). A normal p53 gene was significantly associated with radical resection (P < .004) and survival advantage (P = .02). CONCLUSION: This is the second clinical evaluation demonstrating a significant relation between p53 genotype and response to induction therapy in non-small cell lung cancer. We conclude that the p53 genotype should be evaluated as a predictive marker for response to induction therapy in prospective randomized protocols.

Efficiency of the revised Bethesda guidelines (2003) for the detection of mutations in mismatch repair genes in Austrian HNPCC patients.

The clinical diagnosis of hereditary nonpolyposis colorectal cancer (HNPCC) is based on the Amsterdam II criteria (ACII). The purpose of using the Bethesda guidelines (BG) is to select tumours for microsatellite analysis. Recently, the modified Amsterdam criteria (ACmod) and Bethesda guidelines (BGmod) were proposed to simplify definitions. We evaluated the efficiency of the ACmod and BGmod to identify patients with germ-line mutations in MLH1 and MSH2 in 81 unrelated Austrian HNPCC families. Microsatellite (MS) analysis was performed in 55 tumours. The new criteria included more families than the old ones: BGmod, n = 81; BG, n = 72; ACmod, n = 52 and ACII, n = 35. The more stringent old criteria tended to show greater positive predictive value for association with a germ-line mutation than the corresponding new criteria: BGmod, 23%; BG, 26%; ACmod, 31% and ACII, 37%. The larger number of patients analysed in the ACmod group resulted in greater sensitivity compared to the ACII. The increased workload for BGmod was not associated with greater sensitivity. Microsatellite instability (MSI) significantly enhanced specificity in all subgroups. We recommend the use of the ACmod criteria to select patients for primary sequence analysis, when microsatellite analysis is not possible. If the BG are used, we suggest that BG be given preference over BGmod, as the former signify a lesser workload.

NORE1B, a candidate tumor suppressor, is epigenetically silenced in human hepatocellular carcinoma.

BACKGROUND/AIMS: In human hepatocellular carcinoma (HCC) the ras-proto-oncogene is rarely mutated. We therefore studied the possible inactivation of the putative tumor-suppressors and ras-associating proteins, NORE1A, NORE1B, and RASSF1A in HCCs by mutation or epigenetic gene silencing through promoter-CpG hypermethylation. METHODS: SSCP-analyses, sequencing, and methylation-specific PCR were performed in 28 fibrotic/cirrhotic livers and 40 HCCs. RESULTS: The sequence of NORE1A/B exhibited no deviations and that of the RASSF1A gene a non-silent polymorphism ( approximately 10% of cases) and a missense mutation (one HCC). Both alterations may affect the growth-inhibiting capability of RASSF1A. Epigenetic inactivation of NORE1B was found in 62% of the HCCs and in hepatocarcinoma-cell lines due to considerable promoter-methylation of the gene. Methylation was detected also for RASSF1A in HCCs and hepatocarcinoma cell-lines. As a result, 97% of the HCCs revealed epigenetic silencing of NORE1B, RASSF1A, or both. In contrast every third fibrotic/cirrhotic liver only exhibited silencing of one or both genes. CONCLUSIONS: The candidate tumor suppressor genes NORE1B and RASSF1A are epigenetically down-regulated alone in at least 62%, or in combination in 97% of the HCCs studied. This indicates a frequent and critical event in hepatocarcinogenesis, which may allow HCCs to subverse growth-control in the presence of an unaltered Ras.

Two closely related forms of UDP-GlcNAc: alpha6-D-mannoside beta1,2-N-acetylglucosaminyltransferase II occur in the clawed frog Xenopus laevis.

UDP-GlcNAc:alpha6-D-mannoside beta1,2-N-acetylglucosaminyltransferase II (GnT II; EC 2.4.1.143) is a medial-Golgi resident enzyme that catalyses an essential step in the biosynthetic pathway leading from high mannose to complex N-linked oligosaccharides. Screening a cDNA library from Xenopus laevis ovary with a human GnT II DNA probe resulted in the isolation of two cDNA clones encoding two closely related GnT II isoenzymes, GnT II-A and GnT II-B. Analysis of the corresponding genomic DNAs revealed that the open reading frame of both X. laevis GnT II genes resides within a single exon. The GnT II-A gene was found to be transcriptionally active in all X. laevis tissues tested. In contrast, expression of the GnT II-B gene was detected only in a limited number of tissues. Both GnT II-A and GnT II-B exhibit a type II transmembrane protein topology with a putative N-terminal cytoplasmic tail of 9 amino acids followed by a transmembrane domain of 18 residues, and a C-terminal luminal domain of 405 residues. The two proteins differ at 28 amino acid positions within their luminal regions. Heterologous expression of soluble forms of the enzymes in insect cells showed that GnT II-A and GnT II-B are both catalytically active and exhibit similar specific activities. Both recombinant proteins are modified with N-linked oligosaccharides. N-terminal deletion studies demonstrated that the first 49 amino acid residues are not essential for proper folding and enzymatic activity of X. laevis GnT II.

Expression of the lung resistance-related protein in human and rat hepatocarcinogenesis.

Lung resistance-related protein (LRP) plays an important role in chemoresistance of tumor cells probably by altering nuclear-cytoplasmic transport processes. We analyzed the association between LRP expression and hepatocarcinogenesis in humans and rats by RT-PCR, immunoblotting, and immunohistochemistry. LRP was found in hepatocytes and bile epithelia of normal human and rat liver showing distinct interindividual variations. In human tissues, the LRP expression levels of dysplastic liver nodules, hepatocellular adenomas, and carcinomas were highly variable, including decreased but also distinctly increased staining intensities. Mean expression levels, however, were comparable to the surrounding tissue. Considerable levels of LRP mRNA and protein were also found in human hepatoma cell lines. To study LRP expression from the beginning of hepatocarcinogenesis onward, rats were subjected to a tumor initiation/promotion protocol leading to preneoplastic hepatocytes present as single cells or multicellular clones, followed by adenoma and carcinoma. All of the (pre)neoplastic rat liver lesions expressed, comparable to the surrounding tissue, considerable amounts of LRP. We conclude that LRP might be one mechanism involved in the intrinsically high but variable chemoresistance of normal and (pre)neoplastic hepatocytes.

TP53 genotype but not p53 immunohistochemical result predicts response to preoperative short-term radiotherapy in rectal cancer.

OBJECTIVE: To evaluate and compare the predictive power of p53 gene analysis versus p53 immunohistochemical staining in terms of response to preoperative short-term radiotherapy using 25 Gy in operable rectal cancer. SUMMARY BACKGROUND DATA: Recent studies show that p53 may be a determinant of radiosensitivity being required for induction of apoptosis in case of radiation-induced DNA damage. METHODS: Preirradiation biopsy samples of 64 patients with rectal carcinoma were analyzed. Genetic alterations of the p53 gene were detected by complete direct sequencing of exons 2 to 10. Expression of the nuclear phosphoprotein p53 was assessed by immunohistochemical staining. Results were correlated with histopathology of resected specimens and follow-up data, respectively. RESULTS: Mutations of the p53 gene were present in 45% of tumors. Patients with a normal p53 gene had a significant survival advantage. Comparing pre- and postradiotherapy T category, a reduction was seen in patients with normal p53 genotype only. A mutant p53 genotype was highly specific in indicating stable disease concerning T category after irradiation. Protein overexpression was detected in 61%. Overexpression of the p53 protein was not related to survival or response. The concordance between immunohistochemistry and sequencing was only 0.51. CONCLUSIONS: The authors show that downstaging after short-term radiation may occur but is seen in tumors with normal p53 gene only. Moreover, p53 genotype but not p53 immunohistochemistry is predictive for response to preoperative short-term radiotherapy and patient survival.